Development of a highly polymorphic chloroplast SSR set in Abies grandis with transferability to other conifer species—A promising toolkit for gene flow investigations

Abstract The genus Abies is widely distributed across the world and is of high importance for forestry. Since chloroplasts are usually uniparentally inherited, they are an important tool for specific scientific issues like gene flow, parentage, migration and, in general, evolutionary analysis. Established genetic markers for organelles in conifers are rather limited to RFLP markers, which are more labour and time intensive, compared with SSR markers. Using QUIAGEN CLC Workbench 23.03, we aligned two chloroplast genomes from different Abies species (NCBI accessions: NC_039581, NC_042778, NC_039582, NC_042410, NC_035067, NC_062889, NC_042775, NC_057314, NC_041464, MH706706, MH047653 and MH510244) to identify potential SSR candidates. Further selection and development of forward and reverse primers was performed using the NCBI Primer Blast Server application. In this article, we introduce a remarkably polymorphic SSR marker set for various Abies species, which can be useful for other conifer genera, such as Cedrus, Pinus, Pseudotsuga or Picea. In total, 17 cpSSRs showed reliable amplification and polymorphisms in A. grandis with a total of 68 haplotypes detected. All 17 cpSSRs amplified in the tested Abies spp. In the other tested species, except for Taxus baccata, at least one primer was polymorphic.

and mitochondria the maternal linen (Isoda et al., 2000;Vendramin & Ziegenhagen, 1997;Ziegenhagen et al., 1995).However, all conifer organelle genomes have in common, that their genomes´ silent mutation rates are much lower compared to nuclear sequences (Drouin et al., 2008;Wolfe et al., 1987).Hence, they tend to be more conservative compared with nuclear markers.This combined with the inheritance pathway makes genetic markers in organelles well suitable for gene flow, parentage, migration and, in general, evolutionary analysis.Established conifer chloroplast markers often use restriction enzymes to detect different alleles.However, such RFLP markers tend to be more labour intensive and less polymorphic compared to SSR and SNP markers (Powell et al., 1996).
In this publication, we aligned 12 chloroplast genomes of nine different Abies species to detect regions of variation within the genus Abies.Subsequently, we designed and tested chloroplast SSR primer pairs for promising loci aiming for a polymorphic cpSSR set in Abies grandis.Primer candidates were tested in an A. grandis test sample set, which covers the complete native range of the species, as well as additional conifer species.In the present article, we introduce a highly polymorphic, easy-to-use cpSSR marker set for A. grandis and additional conifer species.

| SSR identification and primer design
We aligned 12 chloroplast genomes of different Abies species (Table 1) using the QIAGEN CLC Workbench 23.03 (Qiagen, Hilden, Germany) to detect chloroplast DNA variation in the Abies genus.All sequences originate from the NCBI database (Sayers et al., 2021) in FASTA format.
Manual browsing for regions of variation in short repetitive motives in the alignment revealed potential SSR candidates, of which we chose only those, flanked by conservative nucleotide sequences.The NCBI Primer blast server application (Ye et al., 2012) was used to develop forward and reverse primers in highly conservative strand regions around the SSR candidates.Position in the chloroplast genome (Table A1) of the selected (Table 2) polymorphic and the nonpolymorphic rejected (Table A2) markers was determined by blasting against the NCBI database.

| Material
Sample material from A. grandis originates from a sample collection in the German IUFRO provenance trials in Lower Saxony, Hesse and North Rhine Westphalian.In total, 96 A. grandis individuals from all seed provenance regions (Rau et al., 2008) in the natural distribution range were included (Table A3).Sample material for other co- KG (for details see Table A4).Also, 96 samples including 12 Abies spp., 2 Cederus spp., Larix decidua, Picea abies, Pinus sylvestris, Pseudotsuga menziesii and Taxus baccata were used in the transferability tests.

| DNA extraction
From all fresh sample, genomic DNA was extracted from mature needle tissue.We used approximately 50 mg fresh or 25 mg dried materials cut into small pieces, frozen in liquid nitrogen and ground in an MM300 ball mill (Retsch, Haan, Germany) for 2 min at 30 Hz.
For the extraction, we used the DNeasyTM 96 Plant Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol.Stored DNA samples were previously extracted in the same way.For P. abies, 5 mL of 26% polyvinylpyrrolidone solution was added to the 90.5 mL lysis buffer during the extraction.

| Primer pair amplification and scoring
Primer testing was done performing PCR in a Biometra TProfessional thermocycler (Analytik Jena, Jena, Germany) with a touchdown protocol, consisting of 15 minutes initial denaturation (95°C), followed by 10 cycles of 60 s of denaturation (94°C), 60 s annealing (60°C; Δ-1°C each cycle) and 60 s extension (72°C).Subsequently, another 25 cycles were performed, using the same temperatures and times, but maintaining 50°C in the annealing step.The protocol was finalized by 20 minutes of extension (72°C).Each volume (15 μL) contained about 2 ng of sample DNA, 1.05 pmol Tris-HCl and 0.263 pmol (NH4)2SO4, 37.5 pmol MgCl2, 2.5 pmol of each dNTP and 1U HOT FIREPol® Taq polymerase (Solis BioDyne, Tartu, Estonia).Polymorphic primer pairs were multiplexed with up to four primer pairs.Each sample volume contained 2.5 pmol M13 Primer (fluorescent dye labelled), 0.5 pmol forward and 1.25 pmol reverse primer of each tested primer pair in the respective multiplex.Each forward primer had an M13-tail addition to the designed sequence.The fragment sizes of the PCR products were determined using an ABI Genetic Analyzer 3130xl (Applied Biosystems, Foster City, CA, United States) and the accompanying Genemapper software v3.7 (Applied Biosystems, Foster City, CA, United States) (Figure A2).
Initial tests for successful amplification, fragment polymorphisms and good delimitation between fragment sizes were assessed using 16 samples of four distinct seed provenances.Subsequently, the polymorphic and well-distinguishable primer pairs were tested in an enlarged data set of 96 A. grandis samples across the distribution range to estimate the degree of polymorphism (Table A3).
Seed provenance regions follow the classification according to Rau et al. (2008).To test the reproducibility and verify the scoring of the data, an independent PCR, according to the protocol described above, was performed on these 96 samples.Additionally, we tested all polymorphic primer pairs in a sample set composed of 12 different related Abies and seven other conifer species (Table 3/Table A4).

| RE SULTS
We developed 17 highly polymorphic primer pairs, which exhibited up to 10 different alleles in a comparatively small data set of 96 individuals (Table 2).All developed primer pairs amplify fragment sizes between 70 and 450 bp, contain between 36 and 63% Guanine and Adenine (mean ~ 50.1%) and are characterized by low chances of forming hair-pin structures (Table A5).Melting temperatures ranged between 57°C and 61°C for all primers, with less than 2°C difference between primers of the same pair.The majority of the promising SSR candidates (repetitive motifs, polymorphic between different genomes) amplify single base pair repetitive motifs.We selected two double base pair motives, as well.However, only one of those (AGcp15) occur to be polymorphic in the primer tests.Despite the mostly single base pair repeats reproducibility was >99% over all primer in the 96 A. grandis samples in the comparison of the two independently performed PCRs and scorings.
All polymorphic primers amplified in all of the 12 tested Abies species (Table 3).Despite the small sample sizes (2 ≤ n ≤ 8) for each species apart from A. grandis, we observed polymorphisms in the majority of successfully amplified primer pairs across all Abies species.For conifer genera apart from Abies, amplification was often not successful for single primer pairs.However, we observed polymorphic loci in some genera, such as Cedrus, Larix, Picea, Pinus and Pseudotsuga.Only within the Taxus genus none of the primer pairs occurred to be polymorphic.
The haplotype network (Figure A1) shows a group of more closely related haplotypes, including those haplotypes that occur multiple times and in different regions of origin.Within the network, no clear separation of regions based on haplotypes can be observed.

| DISCUSS ION
The Abies balsamea 2 Note: Color code highlights whether loci are polymorphic (green), did successfully amplify (yellow) or did not sufficiently amplify (no color) in the tested species.The number of individuals used per species (N) is indicated in italic.
| 5 of 11 markers provides a valuable addition to the available marker set.Using additional genetic markers enables higher resolution of the genetic diversity, especially in geographically widespread studies.All of those novel cpSSR markers are also well suited for multiplex approaches and might thus contribute to a time and labour effective workflow.
Finally, the transferability of some markers to additional species provides a functional tool for chloroplast haplotype definition in species of other genera, such as Pseudotsuga menziesii, or Picea abies.Certainly, the broad coverage of the distribution range contributed to this high diversity.Moreover, with up to 10 different alleles per primer pair, it is very likely to find even more haplotypes in larger datasets.Together with the option to use the marker set in several different Abies species, its highly polymorphic character makes it a well-suited toolkit for future studies across a wide range of Abies species.The low number of polymorphic cpSSRs in A. balsamea might be due to the fact that only two seeds from the same seed source (Canda, Quebec) were analysed.
The primer pairs AGcp01, AGcp02, AGcp03 and AGcp24 amplified in all tested conifer species and additional primer pairs amplified also in some of the non-Abies species from different conifer genera.However, for very few primer pair-species combinations, the PCR protocol might be not ideal and could be optimized for usage.The majority of the peaks were very clearly recognizable and easy to delimit.Despite their small sample sizes (n ≤ 8), some of the primer pairs, such as AGcp01, AGcp04, AGcp24 and AGcp25, even had various alleles in some tested non-Abies species.Thus, it is likely, that some of the loci exhibit additional alleles in the tested species since our test plate contained only a small sample count of each species.Therefore, primer pairs, which were successfully amplified in the tested species, are promising candidates for those genera.
Despite the predominantly single base pair repeat motifs, the introduced cpSSR set exhibited reliable peaks, which could easily be kept apart and showed >99% reproducibility between repetitions.We chose highly conservative regions to design both, the forward and reverse primers on the upstream and downstream strands.No primers were developed for polymorphic regions in the alignment.SSR primer pairs from such regions would probably not result in reliable results since every possible primer pair would harbour the risk of several SNPs within the binding region.Such polymorphisms could compromise the annealing of the primers, possibly leading to non-amplification of the targeted region in some samples.To examine such high polymorphic gene regions, SNP screening would probably be more promising due to the high abundance of SNPs in this region.However, some of the introduced primer pairs are positioned within gene sequences (Table S1) and, therefore, might be correlated to adaptive traits.Primer pairs excluded after the first test with 16 A. grandis samples could still be of interest in other Abies spp.Comprehensive information about all remaining primer pairs is reported in Table S2.Note: Sample origin refers to the place of sampling or the institution where samples were collected/stored and provenance for known geographic origin of the material.
TA B L E A 5 Self complementarity scores of primers according to NCBI Primer blast server application (Ye et al., 2012).

Primer Self complementarity Self 3′ complementarity
AGcp01-F 3 1 AGcp09-F 4 0 AGcp09-R 6 0 AGcp11-F 4 0 F I G U R E A 1 Median vector haplotype network of the detected 68 variants at 11 cpSSRs in 96 samples of Grand Fir (Abies grandis) across the distribution range.Haplotypes are named numerically with the prefix 'H' and coloured according to the origin of the sample/s containing respective haplotype.Median vectors are displayed as grey circles.Mutation steps are indicated as numbers at and thickness of the connecting edges.Construction of the network was performed with NETWORK 10.2.0.0 (fluxu s-engin eering.com) using the median-joining algorithm (Bandelt et al., 1999).
nifer species stem eighter from a sample collection in the Forest Botanical Garden of the University of Göttingen and the Arboretum in Hørsholm of the University of Copenhagen or were stored DNA samples of projects by the Department of Forest Genetics and tree breeding of the University of Göttingen or by ISOGEN GmbH & Co.

TA B L E 1 NCBI
accession code and according to publication for aligned chloroplast genome sequences from different Abies species.
Primer sequences, repeat motifs, approximate fragment lengths, melting temperatures, GC content, number of observed alleles and polymorphic information content (PIC) in Abies grandis for 17 introduced polymorphic primer pairs.
TA B L E 2 We have developed a reliable and user-friendly cpSSR marker set for the relatively sparsely investigated species A. grandis.In a relatively small sample set, the marker set has already shown remarkable diversity.Further studies building on this marker set will contribute to the understanding of genetic patterns within the natural distribution range of A. grandis.Additionally, we demonstrated the usability of the set in other Abies spp.and conifer genera, improving upon the mostly RFLP-based marker systems available for the chloroplast genome in conifers.Origin of the genetic material of the 96 Abies grandis samples from the different German IUFRO trials.Origin of the genetic material of the 96 samples of other Abies spp.and other conifers.